Vitamin products and methods of obtaining the same



l atentecl Mar. 13, 1951 NITED STATES ATENT ()FFICE VITAMIN PRODUCTS AND METHODS OF OBTAINING THE SAME Joseph J. Pfiffner, Detroit, Mich, assignor to Parke, Davis & Company, Detroit, Mich, a corporation of Michigan N Drawing. Application June 16, 1945, Serial No. 599,955

, pound possessing the chick anti-anemia vitamin activity.

Another object of this present invention is to provide a pure chemical compound obtainable from plant sources possessing the chick antianemia. vitamin activity which is suitable for ad- Claims. (Cl. 260251.5)

ministration to humans or animals and which has little or no growth promoting activity for Ldctobacillus casei but which may be readily converted to the pure chick anti-anemia vitamin having said activity.

It is also an object of the invention to provide steps whereby a pure chemical compound possessing the above properties can be readily isolated from plant or animal sources by economical and novel procedures which lead. to a commercially and therapeutically useful compound.

By plant sources I mean vegetable tissues, such as alfalfa and like plants, or fungi, such as forexample yeast, molds and the like. By animal tissues.l'. mean mammalian tissues containing the chick anti-anemia activity such as kidney, liver, spleen, stomach and like tissues.

It has now been found that the anti-anemia vitamin activity is present in most, if not all, plant and fresh animal tissue sources in a chemical form different from that which it has in autolyzed animal tissues such as liver, spleen, kidney, stomach and the like. This can be proved by comparing the pure chick anti-anemia active products isolated from autolyzed animal tissues with those isolated from plant tissues or fresh animal tissues, as to their property of stimulating growth of bacteria. I have found that the pure chemical'compound possessing chick antianemia activity isolated from plant sources, e. g. yeast, or fresh animal tissue, such as fresh kidney or liver tissue, is essentially inert, so far as its property of stimulating bacterial growth is concerned, although it possesses anti-anemia potency in chicks, while the compound isolated from 2 Y autolyzed animal tissuespossesses both the property of stimulating bacterial growth and the property of exercising an anti-anemia effect in chicks. I have found that plant tissues or fresh animal tissues, their aqueous extracts or concentrates, as well as the pure anti-anemia effective chemical compound obtainable from such plant and animal sources can be subjected to a certain enzymatic digestion, thereby imparting to them the property of stimulating bacterial growth, without destruction of the anti-anemia property. I have for the first time isolated the microbio: logically inactive chemical compound present in plant and fresh animal tissues which is responsible for .the chick anti-anemia potency of said tissues. I have also found that the compound is a relatively simple non-protein, non-carbohydrate conjugate of the above mentioned microbiologically active compound obtainable from I find that good yields autolyzed animal tissue. of the chick anti-anemia vitamin, as obtained from autolyzed animal tissue, are obtainable from plants, fungi and fresh animal tissue by firstisolating the pure conjugated material and then breaking down the conjugated material by enzymatic treatment. The active compound obtained as a result of the enzyme treatment step is the unconjugated material. This same unconjugated material may also be obtained by carrying out the enzymatic treatment on the crude or concentrated conjugate, followed by concentration isolation of the unconjugated material.

and However, for purposes of purity and ease of isolation it is preferable to first obtain the pure conjugate before utilizing the enzymatic treatment. 1

I have also found that the new vitamin c0n-, jugate exhibits physiological properties which are not shown by any of the known vitamins. Although this new vitamin conjugate is effectivein preventing certain types of anemia, in humans and animals, and is obtainable from mammalian liver, it can be shown to be very different from the active principles heretofore separated from liver. One of the outstanding differences between this new'vitamin conjugate and the liver products of the prior art is that the vitamin conjugate is not effective against pernicious anemia whereas the liver products of the prior art are very effective in treatment of this type of anemia.

I have found that the pure chick anti-anemia vitamin conjugate, pteroyl hepta glutamic acid, like the unconjugated vitamin pteroyl glutamic acid, is amphoteric in nature. It forms salts of the metallic elements such as the sodium, potaswhere R is alkyl. I have also found that relatively crude concentrates containing the conjugated anti-anemia vitamin may be esterified with alcohols and the pure vitamin conjugate esters isolated" from the resulting mixture. These latter esters are identical with the esters prepared by direct esterification of the pure vitainiii conjugate itself. Furthermore, the esters of the vitamin conjugate may be hydrolyzed by known methcds'to' the unconjugated vitamin; The esters of the vitamin conjugate form well defined acid addition salts with the strong. minerai" acids such as hydrochloric acid, hydrobromic acid, sulfuric acid and the like acids.

1: have also found that the pure chick antiancmia vitamin conjugate does not contain a protein or. carbohydrate like residue and that its molecular weight is. approximately three times that oi unconjugated anti-anemia vitamin. The ultraviolet absorption spectrum of the purev vitae min'conjugate is very similar to that shown by theunconi ated vitamin itself, difiering only in the heights of the maxima and minima" (Eif values) which would be expected from the increased molecular weight. This indicates that the characteristic ultraviolet absorption spectrum is dependent primarily upon the structure of the portidn of themoleculeresponsible for the chick anti-anemia activity.

"It hasalso been found that the esters of the vitamin conjugate differ from the free vitamin conjugate in thatthey are not convertible to the uhconjug-ated vitamin by enzymatic treatment. However, if the esters are first hydrolyzed to remove the ester radical and the resultant product subjected to enzymatic digestion, the unconjugated anti -anemiavitamin is obtained.

The invention is illustrated by the following examples.

" Example 1' 3.0 lbs. of brewers yeast extract Type III is used. This extract in dry form is a product which can be obtained on the market and is p epared by making fan aqueous extract of yeast cells (Sam chdroniydes cerevisiad) filtering the extract and Y /I\\/N\ 1| i N cirrus-Qo ravda-011203 4 undrmcmom-o-o R enaporating it to dryness. The 30 lbs. of; dryextract." material is dissolved in about 23 gallons of water, the solutionh'eated to the boilingpoint and then filtered through Super-Gel, a diato- Inaceous earth product. Sulfuric acid is. carefully added to' the filtered solution until the. pH i s ;ab out 3;.

4.2 lbs. of. active carbon, such as the product known as Norite SQ-ll, is added to the'acidic sonata; the mixture stirred foran hour, and thehfilteredu The filtrate. is. discardedQflfhe charcoal is washed with water'and then with 4 50% alcohol and the washings, containing various impurities, discarded.

The activity is removed from the washed charcoal by eluting it a few times with 3 gallons of about 5% ammonia dissolved in 50% aqueous ethanol. The eluates are combined and concentrated to 0.65 gallon. The eluates contain 0.65 kilogram of solids and about '75 to 80% of the original activity.

The combined and concentrated eluates are exhaustivel extracted with butyl alcohol at approximately neutral pI-I, thereafter acidified to about pH 3 and again exhaustively extracted with butyl alcohol. The butyl alcohol extracts contain impurities and are discarded in each case.

The extracted aqueous residue containing the conjugate vitamin is freed from butyl alcohol by distillation, and then evaporated to dryness under vacuum at low temperature. About 0.65 lb. of a dry product containing the conjugated vitamin product is thereby obtained.

The dry material is dissolved in 6 liters of water and poured slowly with starring into a mixture of 12.75 liters of acetone and 0.6 liter of 1 N hydrochloric acid. The precipitate which separates is collected and again extracted in the same maner and discarded. The. combined aqueous acetone extracts are designated as solution A. Solution A is treated with about g. of activated carbon, such as the product known as Norite SG-ll, stirred for about an hour and filtered. The filtrate is discarded. The activity is eluted from the charcoal by successively washing with a total of 4 liters of a solution of 5% ammonia dissolved in 50% aqueous ethanol. The. solid is discarded and the combined eluates evaporated to a volume of about 1 liter.

Alternatively, solution A may. be treated with one equivalent, of l N sodium hydroxide solution and the phases allowed to separate. Ihe acetone phase which contains impurities is discarded and the aqueous phase which contains the vita-v min conjugate evaporated to. a volume of about 1 liter.

The concentrated solution obtained by either of the above alternate procedures is shaken with an equal volume of phenol, the layers partitioned and the phenol phase which contains impurities discarded. Thefaqueous phase; which contains the vitamin conjugate is saturated with ammonium sulfate and again extracted with an equal volume of phenol. The. layers are separated and the aqueous layer. discarded.

The phenol layer. containing the activity is shaken with 500 ml. or water and 3 liters of ether, the layers separated and the ether-phenol layer discarded. The aqueous solution isadjusted to pH 7 by the addition of 10% sodium hydroxide solution and 30g. of zinc acetate added. A pre} cipi-tate ofthe zinc salt of. the vitamin conjugate separates which is re moved. The zinc salt is. suspended in 100 ml. of water and the. pH of the mixture adjusted to about 2.5 by the addition of 10% hydrochloric acid. The material which fails to dissolve isdiscarded andthe. aqueous so: lution containing. the vitamin conjugate extracted. with an equal volume of phenol. The aqueous. layer whichcontains impurities is, discard'ed. The phenol layer is shaken with 500 ml.

e 'of water and 3 liters of ether, the layers separated to about 3 by the addition of hydrochloric and the phenol-ether layer discarded. The aqueacid and the mixture chilled. The freevitamin ous solution containing the vitamin conjugateis conjugate which separates as a yellow powder adjusted to pH '7 with 10% sodium hydroxide sois removed by filtration and washed with small lution. The small precipitate which separates is volumes of ice water. The crude product is puri-v removed, discarded and the aqueous filtrate fied by recrystallization from hot water to which evaporated 'to a volume of about 30 ml. on the enough hydrochloric acid hasbeen added to make steam'cone 'at atmospheric pressure or, if prethe pH about 3, washed with alcohol and ether ferred, under reduced pressure. and dried.

- The aqueous solution is treated with g. of w The pure vitamin conjugate is obtained 'as zinc acetate and the zinc salt of the vitamin conyellow, microcrystalline, birefringent spherules jugate which separates removed and dried. The having no melting point but discoloring about aqueous solution is discarded. The dry zinc salt 200 C., partially melting at 230-260 C'. but not is stirred with 50 ml. 'of. 1% hydrochloric acid in melting completely up to 360 C.

methanol overnight. The reaction mixture is 15 The pure vitamin conjugate contains the eleconcentrated to a volume of about 5 ml., taken ments carbon, hydrogen, oxygen, and nitrogen. up in 40 m1. of water, the pH of the mixture is It is pteroyl hepta glutamic acid having the adjusted to about 3 and the mixture placed in formula,

the refrigerator overnight. The first crop of the A sample analysis of the pure crystalline vitamin yellow crystalline methyl ester, 50 mg, is removed conjugate is, by filtration and the filtrate allowed to stand 4 Per cent whereby a second crop of about 20 mg. of the Carbon e 49.6 methyl ester is obtained. Hydrogen 5.4 The crude methyl ester is purified by washing Nitrogen 14.8 with water followed by recrystallization from Oxygen 40.2

50% mehanol' then from 95% ethanol and The pure conjugate has a characteristic ultrafinauy by mcryfstamzatmn f Water After violet'absorption spectrum. The spectrum at pH washin the purified ester with alcohol and then 11 exhibits three absorption maxima at Wave With ether it is dried in the air. The pure ester lengths 365 m 2 5 m and 365 m with is obtained as yellow, microcrystalline, bire: fringent spherules which melt at 212-15 C. with ff decomposition of approximately 77.5, 210 and 214 respectively. The pure methyl ester conta s the elements At pH 3.02 it shows a. maximum at wave length carbon, hydrogen, nitrogen and oxygen. It is 40 2825 with g octamethyl pteroyl hepta glutamide having the Em formula,

Sample analysis of the pure crystalline ester are, of 204 while at pH 1 the maximum is at wave length 297.5 where Carbon per cent 50.9 50.9

Hydrogen do 6.0 6.1 Bi? V Nitrogen (Dumas) has a value of 169. The spectrum a pH 11 shows Oxygen three absorption minima at wave lengths 235 mp, The methyl ester of the vitamin conjugate .ex- 267 ma and 332 m with hibits basic properties and forms acid addition salts with strong mineral acids. For example, the hydrochloride salt is prepared in the following manner. 20 mg. of the pure methyl ester of the vitamin conjugate is taken up in a small amount 267 and 2 W1th v of anhydrous methanol and an excess of 10% g V a hydrochloric acid added to the solution. Acetone is added which causes the hydrochloride salt to fi i fg z ggg g gi g gf w shwn separate. The salt is collected, washed with ace.- E

tone anddried whereby a white microcrystalline v I of 9'7. This ultraviolet absorption spectrum is powder is obtained. H

essentially the same as that shown by the free of approximately 133; 186 and 55 respectively. At pH 3.04 one minimum at wave length 253 III/1.,

Example 2 t vitamin as described in the co-pending applica- 50 mg. of the purified methyl ester of the tion of Joseph J. Pfiffner et al.,serial No. 477,99 vitamin conjugate (prepared as described in Exfiled March 4; 1943, now Patent No. 2,407,096,

ample 1) is suspended in 20 ml. of 0.05 N sodium issued September 3, 1946, but the maxima are hydroxide and the mixture allowed to stand overabout one-third as high, which indicates that the night. The mixture is concentrated by evaporaultraviolet absorption spectrum depends primartion in vacuo, the pH of the concentrate adjusted ily on the structure of the free vitamin itself chemically: combined in. the; vitamin coniu'sateand; that:.the vita-mineportioni isv ahout; one third; of? the vitamin; conjugate: molecule;

The vitamin: activeipart. containedin thegvita min: conjugate= may: also-the deter-mineda bx: split:- ting; the: vitamin. conjugate by;- enzymat c tr at.- ment: and isolating: the: free: vitamin as: described in: Example i: below; That. the; vitamin; conga, tained in the vitamin conjugate is abQufiIOIl thirdt of; the entire; vitamin. conjug t m lec is; alsm shown. by: the calculations based on the assay; of; the; antiranemia. prope-nties. ot the vita min conjugate; chicks-., I

Th pure coniueate;is;non-ni1otein; i natureand. gives: a. negative: biuret. test. The; conjugate ives a; neeativeeMoliseh; test f r arbohydratessor sugars, Ad itional evid ceof. the non, protein nature of the vitamin'conjugateis; that it is dialyzable through cellophane No. 300, is not precipitated by heat in acid solution, by saturated ammonium sulfate at pHlevels between 3 and '7; nor bytrichloroaceticacid.

The pure vitamin conjugate has very little or no growth eiTect on either Lactobacillus casei or Streptococcus Zactus.

The vitamin conjugate is amphoteric, forming salts with alkaline reagents, esters with alcohols and salts with mineral acids.

Some examples of the salts which mayv bepree pared from the vitamin conjugate and alkaline reagents such as alkali or alkaline earth hydroxe ides,v carbonates and the like are the barium (described below in Example 3%, sodium, potassium, lithium, and: calcium salts. The sodium salt is prepared, forexam-plc; in the following manner. 25 mg. of the" pure vitamin conjugate is dissolved in distilled Water and to; the resulting solution a slight excess of l. N. sodium hydroxide added. The solution is concentrated by evaporation in vacuo to a volume of about 3 ml. and sodium salt precipitated by adding acetone. The salt is removed by iiltration,.wa'shed Well with acetone and dried. This salt may also be prepared by adding exactly an equivalent amount of l N sodium hydroxide solution, to a solution of the pure vitamin conjugate in water, freezing the, solution. of the pure vitamin conjugate and subliming the ice therefrom under reduced pressures and at low temperatures. The sodium salts obtained by the two methods are identical;

Vitamin conjugate salts of metalswhich are water insoluble may be prepared by displacement of acetic acid from its salts. Some examples of these; salts are, the zinc, lead, silver, ferric and like salts. These salts may; be prepared as shown in Example 1 by treatment of an aqueous solution of the vitamin conjugat with the corresponding acetate salt of; the metal. These saltsmay also be vpreparedby treatment. of aqueous solutionsof the vitamin conjugate with other soluble metal salts such as. silver nitrate, zinc nitrate, zinc chloride, ferric chloride and the like.

The esters Of the vitamin-conjugate are pres pared by treatment of the; conjugate With an excess of the alcohol and a small amount of a mineral acid such as hydrochloric or sulfuric acids. After standing overnight the mixture is concentrated, diluted-with water and the pI-I of the mixture adjusted to ao'0ut'3. On cooling and standingthe crystalline es'ter'separates. The estei is removed by filtration, washed with waterandrecrystallized from 50% methanol; Some examples of the esters which maybe prepared in this manner are, the methyl, ethyl; propyl, n-Joutyl, iso-butyl, n-amylan'd n-hexyl esters}. The-methylestfi orep re i x. hisnr e dure, Em lie-Pu s vitamin conjugate is, identical with the, methylv ester obtaineddnfixample -l The acid. addition. salts: of; the; vitamin. con-ju.

sate re-b nared ad i an X s i e a Example 3 30 lbs. of brewers yeast extract Type III is used. This extract in dry form is a product which can be obtained on the market and is prepared by making an aqueous extract of yeast cells (Saccharomyces cercm'siae), filtering the extract and evaporatingit to: dryness. TheBO lbs, of= ex: tractmaterial is dissolved in about 23; gallonspfwater, the. solution heated; to the boiling point and then filtered: through Super-Gel, a diatoms..- ceous earth product. Sulfuric acid is carefully added to the filtered; solution until the. pH; is about 35.

422 lbs. of. active carbon, such asthe product known as. Norite. SG:-l:l*i$1 added, to the acidic solution, the mixture stirred for an hour, and then filtered. The filtrate; is. discarded; The charcoal: is Washed with: Water and; then with 551% alcohol-and the washings containing; various impurities, discarded.

The activity is removed. from the washedcharcoal" byelu-ting it; a few times with 3-, gallons oi about. 5% ammonia dissolved. in 59% aqueous ethanol. The eluates are combined and; cone centrated to (3.65 gallon. The eluates contain 7 0.65 kilogram of solids and about 75"to 80%. of

' the original activity.

The comb-inedand concentrated eluates are exhaustively extracted with amyl alcohol at approximately neutral pH, thereafter acidified to about pH 3 and again exhaustively extracted with arnyl alcohol; The amyl alcoholextr-acts con in impur i an are discarded insec case- T e ext acted aqueou r sidue. co i e the c j a vita is. tread from am .hb i atio an then evaporated to dryn s and at low temperature. About 0.65 lb. oi a dry. product containing the; conjugated vitamin p oduct is the b ed.

The dry material is dissolved, in 6 liters of water and poured slowly with stirring into a mix-- ture of 12.75 liters ofacetoneand 6.6 liter of l N hydrqchloricacid. The precipitate which separates is collected and again extracted in the same manner and discarded. The combined aqueous acetone extracts are designated as solu- 10.11 a olu on A 5 ea ed t 'eb t 0a of activated carbon, such as the product known as Norite SG-ll, stirred for about an hour and filtered. The filtrate is discarded. The activity is eluted from the charcoal by successively washing with a totalcf elitersof a solution of 5% ammonia dissolved in 5fl% aqueousethanol. The solid-is discarded andthe combined eluates evape oi'ated tea volumeofrabout 1 liter.

' Alternatively, solution A maybe treatedwith one equiealentof" l N sodium hydroxide solution and the phases allowed to separate. The acetone phase which contains impurities is discarded and the aqueous phase which contains the vitamin conjugate evaporated to a volume of about 1 liter.

The concentrated solution obtained by either of the above alternate procedures is shaken with an equal volume of phenol, the layers partitioned and the phenol phase which contains impurities discarded. The aqueous phase which contains the vitamin conjugate is saturated with ammonium sulfate and again extracted with an equal volume of phenol. The layers are separated and the aqueous layer discarded.

The phenol layer containing the activity is shaken with 500 ml. of water and 3 liters of ether, the layers separated and theether-phenol layer discarded. The aqueous solution is adjusted to pH 7 by the addition of 10% sodium hydroxide solution and 30 g. of 'zinc acetate added. A precipitate of the zinc salt'of the vitamin conjugate separates which is removed. The zinc salt is suspended in 100 m1. of water and the pH of the mixture adjusted to'about 2.5 by the addition of 10% hydrochloric acid. The material which fails to dissolve is discarded and the aqueous solution containing the vitamin conju gate extracted with an equal volume of phenol. The aqueous layer which contains impurities is discarded. ihe phenol layer is shaken with 500 ml. of water and 3 liters of ether, the layers separated and the phenol-ether layer discarded. lhe aqueous solution containing the vitamin conjugate is adjusted to pH 7 with 10% sodium hydroxide solution. The small precipitate which separates is removed, discarded and the aqueous filtrate evaporated to a volume of about 30 m1. on the steam cone at atmospheric pressure or, if preferred, under reduced pressure.

1.5 g. of barium hydroxide octahydrate is added to the aqueous solution with stirring and the copious precipitate which separates removed by filtration and discarded. The aqueous filtrate is added with stirring to 10 volumes of methanol which causes the barium salt of the vitamin conjugate to separate. The barium salt is collected, dissolved in 75 ml. of distilled water, the pH of the solution adjusted to about with hydrochloric acid and the solution chilled in the refrigerator. The precipitate which separates is removed and discarded.

The aqueous acidified filtrate is evaporated to a volume of about 40 ml., chilled thoroughly and the small precipitate which forms removed by centrifuging and discarded. The aqueous solution is adjusted to about pH 2.8 with dilute hydrochloric acid and placed in the refrigerator overnight. The yellow vitamin conjugate which separates as microcrystalline, birefringent spherules is removed by filtration. washed with small volumes of cold water and dried; yield mg. If desired, the product may be purified further by recrystallization from hot water acidified to pH 3 withhydrochloric acid.

An additional 15 mg. or the crystalline vitamin conjugate may be obtained by reworking the precipitate resulting from solution of the barium salt of the vitamin conjugate and the subsequent precipitates containing impurities.

The pure vitamin conjugate, pteroyl hepta glutamic acid, obtained by this procedure is identical in all its chemical, physical and physiological properties to the vitamin conjugate prepared as described in Example 2.

I have found that numerous variations may be made in my new processes, shown inthe above examples, for the isolation of the anti-anemia vitamin conjugate. For example, I may omit the extraction of the crude extracts, obtained by elution of the activity from charcoal with ammonia in alcohol, with an alcohol such as butyl or amyl alcohol. If this step is omitted I find that it is advantageous to purify the vitamin conjugate through the methyl ester, as shown in Example 1, and to reprecipitate the conjugate several times as the zinc salt.

Another variation which I have found may be made in the above processes is the omission of the extractions with phenol. In this process it is advisable to purify the vitamin conjugate through the methyl ester, as shown in Example 1, hydrolyze the methylester to the vitamin conjugate according to the process described in Example 2, and then reprecipitate the free acidic vitamin conjugate as the zinc salt several times. The zinc salt is decomposed and the crude vitamin conjugate re-esterified with methyl alcohol and the methyl ester recrystallized until pure. The methyl ester is then again hydrolyzed as shown. in Example 2 to the free vitamin conjugate.

Still another variation which I may make in my new isolation processes described above is the treatment of the original crude aqueous yeast extract with acidified acetone to obtain an extract similar to that of solution A in Examples 1 and 3. The isolation of the free vitamin conjugate may then be accomplished as shown for the purification of solution A in Example 3 or in the combination of Examples 1 and 2.

I have found that the combined process described in Examples 1 and 2 gives high yields of the new vitamin conjugate. This particular process, also yields a product which is more readily purified and obtained in crystalline form while theprocess described in Example 3 gives a product which is also readily purified and obtained in crystalline form butthe yield is slightly lower.

In the above examples I have shown the isolation of the new vitamin conjugate from yeast for the purpose of comparing the yields of the product as isolated, from the same source by difierent processes; However, it should be understood that I may also isolatethe vitamin conjugate from other plant and .animaltissues by application of the processes which I have described above. For examplewhen using fresh beef kidney as a source of the new vitamin conjugate I proceed as follows: lbs. of ground'fresh beef kidney is extracted with about 40 gallons of water and the extracts heated to boiling for 15 minutes. I find that boiling theextracts is necessary to insure destructionof the enzyme present in the tissue which on prolonged digestion at normal temperatures or on autolysis of the tissue splits the vitamin conjugate to yield the acidproduct described and claimed in Patent No. 2,407,096 above referred to. The aqueous extracts are filtered, cooled and treated by any of the various processes described above.

that will either cure or prevent anemia. Under these conditions a curative unit is defined as the least amount of the test substance, given in 6 Grams Casein'purified 25.00 Casein purified "biotin conc 5.00 Cornstarch 36:00 Lard "16.85 Salts (O'and U) 3.90 M'I1SO4AH2O 0.1 Cellufiour 3;!) Gelatin 1010 Vitamin .mixture '(ADEK)" '0;1 5 Vitamin "mixture f(B 'complex') 0.1'0 Choline hydrochloride 0120 Pantothenic acid 0501 Vitamins per 100 grams of ration:

. 20 micrograms of "biotin 320International Units o'f vitamin A 32 international Units of :vitamin'D 10 milligrams of 2 -mthyl-l ,4-nap'hthoquinone '4 milligrams of a-tocopherol 3. "Thiamine, 0.4 milligram Riboflavin, :4 milligram Pyridoxin, 0.6 milligram I Inositol, 50;0milligrams 'Para-am'inobenzoic acid, 15.0 milligrams Nicotiriic ac'id, '0;5 milligram Z'Jlhe .pure crystalline chickantieanemia vitamin conjugate, prepared .as -.described i-in .Examples .2 and .3, when assayed according to the above ,procedurein chicks fed on the. anemia producing diet described has one curative unit per 'fiogamma and .one prophylactic unit per 10.45 gamma. The crystalline .methfl ester of thepure vitamin conjugate, prepared as described in Example v.1, .like- Wise has about .one curative unit per 60 .gamma and about .one prophylactic unitper 0.45 gamma. The pure chick anti-anemia vitamin, prepared as described in Example 4 and described and claimediin Patent No.'2,40.'7,096 above referred to, when assayed by this method in chicks fed on the above anemia producing diet has one curative,

unit ,per 20 ,gamma and one prophylactic unit per 0.15 gamma.

Example 4 2001mg. "of the pure vitamin conj ugate, pteroyl "hepta :g'lutamic acid, prepared as described in "Example 2 or 3 above .is taken up in '"a small amount of water anfd'200 ml. o'f'anaqueous 'extract "of -fresh .kidney added and {the "mixture 'digested'for 'a day or two at 37 C. The kidney extract is prepared by grinding "100 g. of'fresh' with an acidified organic solvent,.:such asibu'tyl alcohol which is acidified With mineral acid to a pH less ithan' about 14,:say 13333. The acidified solvent substantially:completely extracts'itheiunconjugated vitamin from the :digestion "mixture.

The butanol:solution 'of th'eivitamin :concen 1 agueous filtrate containing the barium salt of the vitamin is then treated'with a soluble zinc :salt, such asizinc acetate, in order to precipitate the less soluble zinc-salt :of the vitamin. The zine salt is removed by filtration :and converted to :the

soluble ammoniumsalt byrsuspending the;salt:in

water and treating themixture with ."ammonium oxalate solution. .The insoluble :zinc oxalate which ;precipitates.is removed by filtration and the filtrate lmaide slightly :acidic. The :free vita- .min which separates is filtered oil and dried.

.Theiree vitamin acid, pteroyl 'glutamic a1cid,:prepared in :this :manner .is substantially pure. A slightly higher purity may be obtained by :recrystallization of the vitamin from water :or methanol or .-mixtures of the two *solvents.

f-This pure vitamin acid, -pteroyl glutamic acid, is identical with the 'acid product {described and claimed in Patent No. 2,407,906, issued September 3, 1946.

.I "claim:

.1, Process for the isolation of :a compound having :chick anti-anemia activity and substantially no .growth effect on FLQCtObGCiZZZLS casci Which comprises adsorbing :saicl activity on :charcoal from an acidified aqueous extract :ccn- .taining the chick anti-anemia activity, eluting the activity with :aqueous alcoholic ammonia. concentrating the eluate andextracting the {00ncentratezexhaustively with a lower'aliphatic 1alcohol having a :slight 'miscibility with water, evaporating the aqueous phase containing the'desiredactivity to dryness, taxing the residue up inwater, adding the resultant solution tea-lower alkyl ketone acidified with 'mineral acid, :filtering the mixture, adsorbing the activity vfrom the filtrate on charcoal, eluting the activity with aqueous alcoholic ammonia, concentrating the eluate, extractingrthe concentrate with phenol, saturating the .-aqueous phase containing the chick anti-anemia activity with ammonium 4sulfate, extracting :the solution with :phenol, treating .the phenol phase with ether and Water, .adlusting the ,pH of the aqueous phase which separates to about '7, precipitating the :active :principle from :said aqueous phase as rthe.:zinc .salt, suspending th zinc saltin :an aqueous acid solution :at :about apI-I 25, filtering "the mixture, extracting :the filtrate with phenol, treating the phenol phase with ether and watc -adjusting the pH of the resultant aqueous phase to about 7, concentrating "the solution, precipitating the :acvtivetpr'inciple sfrom the concentrate as the :zinc salt, treating the dry 'zinc salt with lower alkyl alcohol and a small amount of mineral :acid, evaporating the mixture, adding water :and sepmating the precipitated crystalline lower alkyl ester of the-chick anti-anemia factor, hydrolyzingtthe loweralkyl ester with alkali and separating from the hydrolysate in substantially pure form the vitamin conjugate having chick anti- 13 anemia vitamin activity and substantially no growth eiiect on Lactobacillus casei, 2. Process for the isolation of a compoun having chick anti-anemia activity and substantially no growth effect on Lactobocz'llus casei which comprises adsorbing said activity on charcoal from an acidified aqueous extract containing the chick anti-anemia activity, eluting the activity with aqueous alcoholic ammonia, concentrating the eluate and extracting the concentrate exhaustively with a lower aliphatic alcohol having a slight miscibility with water, evaporating the aqueous phase containing the desired activity to dryness, taking the residue up in water, adding the resultant solution to a lower alkyl ketone acidified with mineral acid, filtering the mixture, treating the filtrate with one equivalent of dilute alkali, concentrating the eluate, extracting the concentrate with phenol, saturating the aqueous phase containing the chick anti-anemia activity with ammonium. sulfate, extracting the solution with phenol, treating the phenol phase with ether and water, adjusting the pH of the aqueous phase which separates to about '7, precipitating the active principle from said aqueous phase as the zinc salt, suspending the zinc salt in an aqueous acid solution at about pH 2.5, filtering the mixture, extracting the filtrate with phenol, treating the phenol phase with ether and water, adjusting the pH of the resulting aqueous phase to about 7, concentrating the solution, precipitating the active principle from the concentrate as the zinc salt, treating the dry salt with lower alkyl alcohol and a small amount of mineral acid, evaporating the mixture, adding water and separating the precipitated crystalline lower alkyl ester of the chick anti-anemia factor, hydrolyzing the lower alkyl ester with alkali and separating from the hydrolysate in substantially pure form the vitamin conjugate having chick anti-anemia vitamin activity and substantially no growth effect on Lacz'obaczllus cascz'.

the carboxylic acid vitamin conjugate, treating the dry zinc salt with lower alkyl alcohol and small amount of mineral acid, evaporating the mixture, adding, water and separating the precipitated crystalline lower alkyl ester of the chick anti-anemia factor, hydrolyzing the lower alkyl ester with alkali and separating from the hydrolysate in more highly purified form the vitamin conjugate having chick anti-anemia vitamin activity and substantially no growth efiect on Lactobacillus casei. Y

4. Process for the isolation of a compound having chick anti-anemia activity and substantially no growth effect on Lactobacillus cascz' which comprises adsorbing said activity on charcoal from an acidified aqueous extract contain ing'chick anti-anemia activity, eluting the activity with aqueous alcoholic ammonia, concentrating the eluate and extracting the concentrate exhaustively with a lower aliphatic alcohol having a slight miscibility with water, evaporating the aqueous phase containing the desired activity to dryness, taking the residue up in water, adding the resultant solution to a lower alkyl ketone acidified with mineral acid, filtering the 14 mixture, adsorbing the activity from the filtrate on charcoal, eluting the activity with aqueous alcoholic ammonia, concentrating the eluate, extracting the concentrate with phenol, saturating the aqueous phase containing the chick anti-anemia activity with ammonium sulfate, extracting the solution with phenol, treating the phenol phase with ether and water, adjusting the pH of the aqueous phase which separates to about '7, precipitating the active principle from said aqueous phase as the zinc salt, suspending the zinc salt in an aqueous acid solution at about pH 2.5, filtering the mixture, extracting the filtrate with phenol, treating the phenol phase with ether and water, adjusting the pHiof the resultant aqueous phase to about '7, concentrating the solution, converting the active principle in the concentrate to the water soluble barium salt with barium hydroxide, adding the solution of the barium salt to a water soluble lower alkyl alcohol, separating the precipitated barium salt therefrom, decomposing said barium salt with mineral acid, filtering the mixture, concentrating the filtrate and separating from the concentrate in substantially pure form the vitamin conjugate having chick'antianemia, vitamin activity and substantially no growth efiect on Lactobacillus casei.

5. Process for the isolation of a compound having chick anti-anemia activity and substantially no growth effect on Lactobacillus casei which comprises adsorbing said activity on charcoal iroman acidified aqueous extract containing the chick anti-anemia activity, eluting the activity with aqueous alcoholic ammonia, concentrating the eluate and extracting the concentratecxhaustively with a lower aliphatic alcohol having a slight miscibility with water, evaporating the aqueous phase containing the desiredactivit; to dryness, taking the residue up in water, adding the resultant solutionto a lower alkyl ketone acidified with mineral acid, filtering the mixture, treating the. filtrate with one equivalent of dilute alkali, concentrating the eluate, extracting the concentrate with phenol, saturating the aqueous phase containing the chick anti-anemia activity with ammonium sulfate, extracting the solution with phenol, treating the phenol phase with ether and water, adjusting the pH of the aqueous phase which separates to about '7, precipitating the active principle from said aqueous phase as, the

. zinc salt, suspending the zinc salt in an aqueous acid solution at about pH 2.5, filtering the mixture, extracting the filtrate with phenol, treating the phenol phase with ether and water, adjusting the pH of the resultant aqueous phase to about 7, concentrating the solution, converting the active principle in the concentrate to the water soluble barium salt with barium hydroxide, adding the solution of the barium salt to a water soluble lower alkyl alcohol, separating the precipitated barium salt therefrom, decomposing said barium salt with mineral acid, filtering the mixture, concentrating the filtrate and separating from the concentrate in substantially pure form the vitamin conjugate having chick anti-anemia 3116 1011 :nharcQal, .eluting the activity with :z-agueous ianti-eanemia Nitamin activity and substantially .zalcoholic ammonia,nconcentiiatingthe'eluate, exno growth efiect ion Lactobacillus cascz'.

tracting the concentrate witlnphenol, saturating 1.8. ,Atpteroylheptaglutamicle compound having .the :aqueous phase :containing the chick l-anti- .theiorrmula,

' OR coon V0 "anemiaiactivity withammonium sulfate;extract- "where-Rrisloweralkyl.

ing thesolution .withphenol, treating the phenol 9. An octamethyl pteroyl hepta giutamide comphase with ether and water, adjusting the -:pH "poundhavingfihe fonnula j p (I) ooocna o leooona (H N CH -NH-t-(-QN T.&HTCHzOHzJJ-)a NHCH+CH2OH2G- OCH3 'ofthejaqueous'p'hasewhich scparates'toabout'l, 10. A'compound of the class consisting of the precipitating theactiveprincipleiromsaitlagueesters and the salts "of the esters of an :organic 'ous phase as the 'zinc "salt, suspending'thezinc "carboxylic acid. vitamin conjugate effective salt in 'an'aqueous acid solution'at'about pH2.5, against anemia of chicks sufiering from a Lie- 'filtering the mixture, extracting the ffiltrate with fici'ency of the anti-anemia vitamin and having phen0l,;tr(-:ating the phenolphase with'eth'er and "substantially -no growth effect on Lactobac'iilus Water, "adjusting the 'pHro'f the resultant aqueous ca-seisaicl esters being octa 'a'lkyl pteroyl glutamphase to about 7, concentrating the'solution,'pre ides, being yellow in color and crystallizing as cipitating the active principle from the concen- 'microcrystallinebirefrigent spherules, said'esters trateasthe zinc salt,-treating'the dry'zinc salt being relatively insoluble in cold water, more with lower-alkyl alcohol anda small amountof soluble in hot Water, soluble in methanol and .mineral acid, evaporating the -mixture, adding ethanol, being-dialyza'ble through cellophaneiiflfl, waterand. separating the precipitated crystalline not precipitated by ammonium sulfate, giving lower'alkyl'ester'of the chick'anti anemia factor, negative reactions in the 'biuret test and trihydrolyzing thellower alkyl'esterwith'alkaliand chloroaceticacid test for'proteins and givinga separating from the hydrolysate-in "substantially =negative'Molisch test for carbohydrates. vpure form the vitamin conjugate having chick JOSEPI-I'J. PFl-FFNER. anti-anemia vitamin activity and substantially REFERENCES CITED :no growthefiect on Lactoba cz'llus cas'ez'. I v

In aprocess for Obtaininglan organic cap in Theziollowmg references are of record'm the -=hoxylic acid'vitamin'conjugate, eiTective against patent: "anemia of chickssuffering from deficiency fof lthe UNITED STATES "PATENTS 'anti-anem'ia'vitamin and having:substantiallyno growth effect on Lactobacillu's "case'i, the steps 333 5 'ii' e 1935 comprising forming from animpurea'queous'ex- 45 tract containing chick antieanemia "activity, an OTHER REFERENCES aqueous solution of the crude barium-salt of.:said "Edgar "Biochem J" VOL 31, pp conjugate,addingthe solution of the barium tsalt 937 Water Soluble lower alkyl'alcoholfseparating Waller ,et al.: Ann. New York Acad. Sci, vol.

"'the'preeipitated barium salt therefrom, decom- 50 .48,1pp 233-287 (1946),

posing saidbarium salt with -mineraliacid,filter- Heinle t Ann New York Acad s V61 ing the mixture, concentrating the filtrate and 48, pp. 343-1346419416).

"separating fmm the cancemrateiwmore highly Welch .et.al.: Ann. New 'York Acad. Sci, vol.

purified form the 'vitamintconjugatehaving chick 4 3 7 349 (1946) 

1. PROCESS FOR THE ISOLATION OF A COMPOUND HAVING CHICK ANTI-ANEMIA ACTIVITY AND SUBSTANTIALLY NO GROWTH EFFECT ON LACTOBACILLUS CASEI WHICH COMPRISES ADSORBING SAID ACTIVITY ON CHARCOAL FROM AN ACIDIFIED AQUEOUS EXTRACT CONTAINING THE CHICK ANTI-ANEMIA ACTIVITY, ELUTING THE ACTIVITY WITH AQUEOUS ALCOHOLIC AMMONIA, CONCENTRATING THE ELUATE AND EXTRACTING THE CONCENTRATE EXHAUSTIVELY WITH A LOWER ALIPHATIC ALCOHOL HAVING A SLIGHT MISCIBILITY WITH WATER, EVAPORATING THE AQUEOUS PHASE CONTAINING THE DESIRED ACTIVITY TO DRYNESS, TAKING THE RESIDUE UP IN WATER, ADDING THE RESULTANT SOLUTION TO A LOWER ALKYL KETONE ACIDIFIED WITH MINERAL ACID, FILTERING THE MIXTURE, ADSORBING THE ACTIVITY FROM THE FILTRATE ON CHARCOAL, ELUTING THE ACTIVITY WITH AQUEOUS ALCOHOLIC AMMONIA, CONCENTRATING THE ELUATE, EXTRACTING THE CONCENTRATE WITH PHENOL, SATURATING THE AQUEOUS PHASE CONTAINING THE CHICK ANTI-ANEMIA ACTIVITY WITH AMMONIUM SULFATE, EXTRACTING THE SOLUTION WITH PHENOL, TREATING THE PHENOL PHASE WITH ETHER AND WATER, ADJUSTING THE PH OF THE AQUEOUS PHASE WHICH SEPARATES TO ABOUT 7, PRECIPITATING THE ACTIVE PRINCIPLE FROM SAID AQUEOUS PHASE AS THE ZINC SALT, SUSPENDING THE ZINC SALT IN AN AQUEOUS ACID SOLUTION AT ABOUT PH 2.5, FILTERING THE MIXTURE, EXTRACTING THE FILTRATE WITH PHENOL, TREATING THE PHENOL PHASE WITH ETHER AND WATER, ADJUSTING THE PH OF THE RESULTANT AQUEOUS PHASE TO ABOUT 7, CONCENTRATING THE SOLUTION, PRECIPITATING THE ACTIVE PRINCIPLE FROM THE CONCENTRATE AS THE ZINC SALT, TREATING THE DRY ZINC SALT WITH LOWER ALKYL ALCOHOL AND A SMALL AMOUNT OF MINERAL ACID, EVAPORATING THE MIXTURE, ADDING WATER AND SEPARATING THE PRECIPITATED CRYSTALLINE LOWER ALKYL ESTER OF THE CHICK ANTI-ANEMIA FACTOR, HYDROLYZING THE LOWER ALKYL ESTER WITH ALKALI AND SEPARATING FROM THE HYDROLYSATE IN SUBSTANTIALLY PURE FORM THE VITAMIN CONJUGATE HAVING CHICK ANTIANEMIA VITAMIN ACTIVITY AND SUBSTANTIALLY NO GROWTH EFFECT ON LACTOBACILLUS CASEI.
 9. AN OCTAMETHYL PTEROYL HEPTA GLUTAMIDE COMPOUND HAVING THE FORMUAL. 